Isolation of proteins involved in posttranscriptional gene silencing and methods of use

ABSTRACT

The present invention includes a method for detecting and isolating sugarcane proteins that interact with the HC-Pro and P1 proteins of SrMV and other proteins involved in gene silencing, particularly in sugarcane. The method uses a two hybrid assay with an HC-Pro, P1, or other silencing-related protein-containing bait protein and a prey protein containing a polypeptide encoded by a DNA molecule in a cDNA library. The method also includes identification of false positives through reverse two-hybrid assays and using in vitro techniques such as farwestern blots or pull down assays where plant physiological conditions may be replicated. Finally, interactions may be confirmed in planta. Some novel proteins used in and discovered using the these methods are also identified. Methods of using viral and plant proteins to regulate silencing in plants such as sugarcane are also discussed.

CLAIM TO PRIOR APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 11/252,080 filed on Oct. 17, 2005 now abandoned and publishedas U.S. 2006 0090217, which is a divisional application of U.S.application Ser. No. 10/226,715 filed on Aug. 23, 2002, published asU.S. 2003 0099984 on May 29, 2003 and issued as U.S. Pat. No. 7,001,739on Feb. 21, 2006, both incorporated by reference herein. Thisapplication also claims priority via the two divisional applicationsabove to U.S. Provisional Patent Application Ser. No. 60/314,863 filedon Aug. 24, 2001 and incorporated by reference herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to sugarcane protein isolationand more particularly to isolation and characterization of proteins thatare involved in posttranscriptional gene silencing. The invention alsoincludes sugarcane and sorghum mosaic virus proteins involved insilencing and the cDNAs which encode them. Finally the inventionincludes use of these cDNAs and proteins to regulate silencing.

BACKGROUND OF THE INVENTION

In the course of evolution organisms have developed a spectrum ofdefense mechanisms that target alien, parasitic elements. The mostspecialized defense system is perhaps the vertebrate immune system,which provides effective protection against a wide range of infectiousmicrobes. In the vertebrate immune system, it is essentially peptidesthat are recognized as non-self and eliminated. Transgenic plant studieshave revealed the existence of another, more ancestral level of defenseresponse. Homology-dependent gene silencing can be viewed as a novel,innate host defense system that is capable of recognizing foreignnucleic acids as non-self and inactivating or removing them from thecell. First recognized in plants and fungi, homology-dependent genesilencing mechanisms have now been shown to operate in a wide range ofeukaryotic organisms. In plants, this type of gene silencing may occurat the level of DNA, by inhibition of transcription, or at the level ofRNA, by enhanced RNA turnover. Both viral RNA and transgenes are subjectto these host surveillance systems, which are only poorly understood atthe molecular level. At the core of both silencing events resides amolecular mechanism that is able to recognize nucleic acid sequencehomology.

Transgene-induced gene silencing in plants was originally described asthe coordinated suppression of transgenes that share sequence similarity(Depicker and Van Montagu, 1997). This phenomenon is most often inducedwhen multiple copies of a transgene are present at a single locus.Silencing not only affects all genes in that locus, i.e. in cis, butalso acts in trans, and additionally down-regulates the expression ofother, unlinked transgene(s). Silencing can also affect the expressionof endogenous genes, provided they have sequence similarity to thesilencing transgene, a phenomenon referred to as cosuppression (Napoliet al., 1990; Van der Krol et al., 1990).

In plants, cases of transgene-induced gene silencing belong to twodifferent mechanistic classes: those that occur at the level oftranscription and those that are due to enhanced RNA turnover.Transcriptional gene silencing (TGS) requires sequence identity in thepromoter region and is associated with methylation and inactivation ofthe promoter sequences of the affected genes (Kumpatla et al., 1998). Inposttranscriptional gene silencing (PTGS), the (trans)genes remainactively transcribed but the steady-state RNA levels are highly reduceddue to sequence-specific RNA degradation. Some instances of PTGS areassociated with DNA methylation located in the transcribed portion ofthe genes (Ingelbrecht et al., 1994; 1999).

The expression level, number and configuration of the integratedtransgenes as well as developmental and environmental factors can allinfluence the occurrence of transgene-induced gene silencing.Importantly, transgene-induced gene silencing in plants is reversible,and in the absence of the silencer locus, expression of endogenous genesor other transgenes can be restored to normal. The changes in geneexpression are therefore not due to irreversible changes in DNA butrather are epigenetic.

PTGS behaves as a non-clonal event and, in agreement with this, it hasbeen shown that a sequence-specific signal is involved in the systemicspread of PTGS (Palauqui et al., 1997; Voinnet and Baulcombe, 1997).These experiments allow differentiation of separate initiation andmaintenance phases in PTGS and further suggest that a molecular systemamplifies the silencing signal during the course of long-distancemovement of PTGS. Mutants that enhance (Dehio and Schell, 1994) orsuppress (Elmayan et al., 1998; Mourrain et al., 2000; Dalmay et al,2000) PTGS have been isolated in Arabidopsis thaliana but only two ofthe corresponding genes have been cloned (Mourrain et al., 2000; Dalmayet al, 2000). One of these has no significant similarity with any knownor putative protein (Mourrain et al., 2000) and the other is similar toa RNA-dependent RNA polymerase (RdRp; Mourrain et al., 2000; Dalmay etal, 2000). Establishment of PTGS in plants requires separatelyidentifiable initiation, spread, and maintenance phases, but theproteins involved in these pathways have not been characterized.

Plant virus studies have greatly contributed to the currentunderstanding of gene silencing in general and PTGS in particular.Applying the concept of pathogen-derived resistance, viral genes wereintroduced into plants and resulted in virus resistant phenotypes. Manyresistance phenotypes do not require the expression of a functionalprotein but are mediated at the level of RNA. It is now an establishedfact that a mechanism similar to PTGS is the underlying molecularmechanism in most of these cases (van den Boogaart et al., 1998).

Posttranscriptional silencing of an endogenous plant gene or transgenecan be triggered by replication of a recombinant virus that carriessequences homologous to these genes (Kumagai et al., 1995; Ruiz et al.,1998). This process involves sequence-specific RNA turnover, similar toPTGS induced by transgenes, hence the term virus-induced gene silencing.Moreover, natural virus infection of non-transgenic plants can induce aresistance mechanism that is strain-specific and targeted against RNA,similar to RNA-mediated resistance induced by (silenced) transgenes(Ratcliff et al., 1997; Covey et al., 1997). Transgene- andvirus-induced gene silencing are collectively described ashomology-dependent gene silencing because these mechanisms all targethomologous nucleic acid sequences. It was proposed thathomology-dependent gene silencing acts as a natural plant defensemechanism against invading DNA or RNA elements (Matzke and Matzke,1998).

The demonstration that plant viral proteins can suppress PTGS providesdirect evidence that PTGS functions as a host defense response in plants(Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau andCarrington, 1998). At least 5 different proteins encoded by unrelatedDNA and RNA viruses of plants have now been shown to act as suppressorsof PTGS in Nicotiana benthamiana. Importantly, the suppressionphenotypes induced by these viral proteins are distinct indicating thatseparate steps of the host PTGS defense system are targeted. Forexample, the potyviral helper-component proteinase (HC-Pro) can reversethe effects of PTGS in tissues that were previously silenced, whereasthe 2b protein of Cucumber mosaic virus only affects initiation of PTGS(Voinnet et al., 1999). Although potyviral HC-Pro by itself issufficient to suppress transgene-induced silencing, it appears that thepotyviral P1 protein can enhance its ability to suppress virus-inducedgene silencing (Anandalakshmi et al., 1998; V. Vance). The discovery ofviral suppressors of silencing phenomena is unique to plants. So far, noanimal or fungal viruses have been shown to suppress PTGS in theseorganisms.

It has been proposed that ‘aberrant’ RNA molecules trigger PTGS inplants (Lindbo et al., 1993). The exact nature of this aberrant RNA isunknown but it could be double-stranded RNA (dsRNA) (Waterhouse et al.,1998), prematurely terminated transcripts, levels of RNA that exceed acertain threshold, or some other unusual characteristic. These RNAmolecules would serve as templates for an RNA-dependent RNA polymerase(RdRp) and lead to the production of short complementary RNAs (cRNA).These cRNAs would then anneal with homologous mRNAs or viral RNAs andthe resulting double-stranded RNA would be degraded by doublestrand-specific RNases. This model accounts for the sequence-specificRNA turnover and several aspects of it are supported by experimentaldata. For example, an RdRp that is induced during viral infection hasbeen cloned in tomato (Schiebel et al., 1998) and small cRNAs haverecently been identified in transgenic plants that display PTGS(Hamilton and Baulcombe, 1999). The identification of a doublestrand-specific RNase in Caenorhabditis elegans and a RdRp-like proteinin Neurospora crassa, and recently in Arabadopsis, as essentialcomponents of PTGS-like mechanisms in these organisms (see below)provides further support for this hypothesis.

RNA-mediated genetic interference (RNAi) in C. elegans is a process thatclosely resembles PTGS in plants: both act at the posttranscriptionallevel and result in sequence-specific RNA turnover (Tabara et al., 1998;Montgomery and Fire, 1998). The trigger for RNAi in C. elegans is wellcharacterized and consists of dsRNA (Sharp, 1999). RNA-specificsilencing can be induced by locally injecting homologous dsRNA moleculesin a few cells. Silencing then spreads from the site of injection intoneighboring cells and tissues and is even transmitted to the F1 progeny.The ability of silencing to move both in space and over time stronglysuggests that amplification of the silencing signal is taking place,similar to PTGS in plants.

Recently, several genes have been identified in C. elegans that arerequired for this interference process. The MUT-7 gene encodes a homologof RNaseD, which is a double strand-specific RNase (Ketting et al.,1999). The RDE-1 gene belongs to a family of genes that are conservedfrom plants to vertebrates and several members of this family arerequired for gene silencing mechanisms in animal systems (Tabara et al.,1999). Interestingly, mutations in both these genes reactivatemobilization of endogenous transposons, suggesting that one function ofRNAi is transposon silencing. Sequence-specific inhibition of genefunction by dsRNA has also been demonstrated in trypanosomes, Drosophilaand planaria and has been used in these organisms as a method todetermine gene functions (Kennerdell and Carthew, 1998; Misquitta andPatterson, 1999; Sanchez Alvarado and Newmark, 1999).

Transgene-induced PTGS is termed ‘quelling’ in the fungus N. crassa(Cogoni and Macino, 1997a). Quelling-defective (qde) mutants of N.crassa, in which transgene-induced gene silencing is impaired, have beenisolated and could be classified in three qde complementation groups(Cogoni and Macino, 1997b). Two QDE genes that belong to two differentcomplementation groups, have recently been cloned. The QDE-1 geneencodes a protein that contains an RdRp-motif (Cogoni and Macino, 1999a)and QDE-3 belongs to the RecQ DNA helicase family (Cogoni and Macino,1999b).

As summarized above, there has been substantial progress in the generalunderstanding of PTGS in plants and its importance as part of a generaldefense system is now fully appreciated. However, all of the biochemicalpathways of PTGS and the enzymes that are involved have not yet beenelucidated in plants. Insight into these mechanisms may come fromanalyzing mutants that are defective in PTGS. This approach has alreadybeen used with success in Neurospora and C. elegans and is currentlyalso being followed for Arabidopsis. While this strategy is relativelystraightforward and will surely result in the identification of genesthat play a central role in this process, there are also limitations.For example, gene redundancies and possibly lethal, loss-of-functionphenotypes might prevent identification of certain genes. There are alsopractical problems in generating and screening a sufficiently largenumber of mutants which limit this approach to model plants such asArabidopsis.

An alternative or complementary approach involves directly identifyingthe host factors that mediate PTGS. The identification of viral proteinsas suppressors of PTGS provides the necessary tools to pursue thisstrategy.

Identification and characterization of proteins that interact with aviral suppressor of PTGS will have an impact on understandingfundamentals of virus-host plant interactions, particularly on themechanisms that plants employ to combat viral infection and on thecounterdefensive strategies that viruses use to suppress or evade theseresponses. To date, viral suppression of PTGS is a process unique toplants. However, because PTGS is a defense mechanism that is conservedamong various eukaryotic kingdoms, the identified protein interactionsmight also shed light on the molecular mechanisms of silencing phenomenain other organisms.

In addition to significance for basic (plant) molecular virology,establishing the biochemical pathways of host defense responses willfacilitate the development of improved virus control strategies inplants. PTGS-based approaches for virus control are already in use butthe lack of a solid understanding of the phenomenon necessitates a moreempirical approach and has an uncertain outcome. Also, such approachesare currently limited because of their narrow range. Possible andrealistic improvements involve enhanced and more predictable triggeringand broadening the scope of the PTGS defense system.

Use of the method of the present invention will also contribute to plantgenetic engineering in general. It is now clear that transgenes inplants (and other organisms) can be perceived as intrusive elements andconsequently are inactivated. Developing procedures that allow stableand predictable transgene expression is one of the challenges of geneticengineering. The monocot crop plants provide the most important sourceof food worldwide and offer great potential for improvement throughgenetic transformation, not only for traits related to food productionbut also as recombinant expression systems for high value products.

Finally, gene silencing can be used as a way to produce ‘knock-out’phenotypes in reverse genetic studies. This has already beensuccessfully applied in animal systems and its potential has beendemonstrated in plants. With an increasing number of genes beingdiscovered in sugarcane, many of which have no known function, it can beexpected that these approaches will become even more important in thefuture.

Thus, the yeast two-hybrid method of the present invention has been usedto unravel the pathway(s) of PTGS and plant defense responses and novel,key proteins involved in this process have been identified. In doing so,a cDNA library from silenced plant tissues rather than non-silence planttissues has been used. These proteins and genes can be applied towardsregulating PTGS of transgenes, endogenous plant genes, and viral genes.Specific applications of the present invention include but are notlimited to, improved strategies for engineered virus resistance,increased expression of transgenes by inhibiting silencing, andmodulation of silencing of native genes to obtain desirable traits or infunctional genomic studies.

SUMMARY OF THE INVENTION

The present invention includes a method of isolating nucleic acidencoding a plant polypeptide active in PTGS. As used throughout theapplication, plant may mean a mature plant, an embryonic callus, orother stages of plant development. It may also mean a portion of aplant, a plant tissue, or a plant cell.

The first step of a method of the above method involves selecting a baitnucleic acid which encodes a bait protein active in PTGS in plants orsuppressive of PTGS in plants. After bait selection, a cDNA prey librarymay be prepared from a plant that actively exhibits PTGS at the time oflibrary generation. If an entire plant exhibiting PTGS is not available,tissues in which PTGS is exhibited may be selected.

After the bait and prey are selected, a yeast two-hybrid assay may beconducted with the bait and prey nucleic acids. Prey cDNA that yields atrue positive yeast two-hybrid assay result encodes a polypeptide activein PTGS in the plant. True positive status may be verified using methodsknown in the art, such as null controls, reversal of bait and prey, andin vitro and in planta studies of interactions. Such in vitro assay mayinclude farwestern blot assays and pull down assays. They may beperformed under plant physiological conditions to eliminate falsenegatives and false positives. In planta studies may be performed in anembryonic callus or other plant tissue.

In an exemplary embodiment of the above method of the present invention,the bait nucleic acid comprises a sequence selected from SEQ.ID.NO. 1,SEQ.ID.NO. 3, SEQ.ID.NO. 5, SEQ.ID.NO. 7, SEQ.ID.NO. 9, or SEQ.ID.NO.11. The entire nucleic acids of these sequences may be used or onlyportions thereof. Additionally, substituted nucleic acids and nucleicacids with similar identities may also be used.

In another exemplary embodiment the plant is a monocot, particularlysugarcane and more particularly Saccharum hybrid cultivar CP72-1210.

The present invention also includes several SrMV and sugarcane novelproteins and nucleic acids. Novel nucleic acids are provided inSEQ.ID.NOS. 1, 3, 5, 7, 9 and 11. Novel amino acid sequences areprovided in SEQ.ID.NOS. 2, 4, 6, 8, 10 and 12. It will be apparent toone skilled in the art that portions of these nucleic acids and proteinsor polypeptides may be used in various applications. Additionally, itwill be apparent to one skilled in the art that nucleic acids andproteins or polypeptides with high similarity, particularly in regionsrelated to functional domains, may also be substituted. The presentinvention includes such variations up to the point of disclosuresalready in the prior art. Each of these proteins is involved in PTGSeither as an activator or suppressor. Some proteins may fail to functionalone and may rather require or assist another protein for theirPTGS-related functions.

The present invention additionally includes transgenic plants includingany of the above novel nucleic acids, proteins or polypeptides. Inparticular, the invention includes a transgenic plant in which PTGS issuppressed that includes the nucleic acid of SEQ.ID.NO. 1 or the proteinof SEQ.ID.NO. 2. It also includes a transgenic plant in which PTGS isenhanced that includes the nucleic acid of SEQ.ID.NO. 3 or the proteinof SEQ.ID.NO. 4.

The invention additionally includes a method of increasing viralresistance in a plant in which a protein suppressive of PTGS in theplant is selected and the plant is transformed with a nucleic acidencoding the PTGS suppressive protein.

Another method of the invention involves a method of increasingexpression of a transgene in a plant by selecting a protein active inPTGS in the plant and transforming the plant with a nucleic acidencoding the protein active in PTGS.

Yet another method of the present invention involves suppressingexpression of a native gene in a plant by preparing a vector including anucleic acid with a sequence of the coding portion of the gene whereinthe nucleic acid, upon transcription, products an mRNA molecule doublestranded in the region corresponding the to the coding portion of thegene. A plant is then transformed with the vector.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention and theadvantages thereof, reference is now made to the following descriptiontaken in conjunction with the accompanying drawings, wherein likereference numbers represent like parts, and which:

FIG. 1A provides the cDNA (SEQ.ID.NO. 1) and FIG. 1B provides the aminoacid (SEQ.ID.NO. 2) sequences for Sorghum Mosaic Virus (SrMV) P1/HC-Proaccording to an embodiment of the present invention;

FIG. 2 is a Northern analysis of SrMV (coat protein) CP mRNA in whichthe lane labeled “S” includes mRNA derived from a plantposttranscriptionally silenced for SrMV CP; lanes labeled “RetransformedS” include mRNA from samples of the silenced plant which wereretransformed with either nptII alone or with nptII and SrMV P1/HC-Pro;the lane including mRNA from a plant that is not silenced is labeled“NS”; and the empty lane is labeled “b”;

FIG. 3 is a β-galactosidase filter lift assay on L40 yeast transformedwith various DNA binding and activation domain constructs and grown onmedia that does not select for interactions (−leu +zeo) or media thatdoes select for transformation and interaction (−leu, −his +zeo); wherethe region labeled “A” represents yeast transformed with SrMV HC-Probait+SrMV HC-Pro prey; the region labeled “B” represents yeasttransformed with SrMV HC-Pro bait+empty prey; the region labeled “C”represents yeast transformed with lamin bait+SrMV HC-Pro prey; and theregion labeled “D” represents yeast transformed with empty bait+SrMVHC-Pro prey; although the figure is presented in black and white, growthin the regions marked A on both plants appears blue while, on the −leu+zeo plate, growth in regions B-D appears red;

FIG. 4 is a gel analysis of the cDNA library cloned in pIVING1154; whereLane 1 contains a λPstI size marker; Lane 2 contains a lower band of 0.8kb which is the CP coding region amplified from the plasmid cDNA libraryand a top band of 6.6-kb which is the plasmid vector; and Lane 3contains a SfiI digest of plasmid library showing the 6.6-kb vector andinserts visible as a smear with size range between 0.5 and 2.0 kb;

FIG. 5 is a β-galactosidase filter lift assay on L40 yeast transformedwith various DNA binding and activation domain constructs and grown onmedia that does not select for interactions (−leu +zeo) or media thatdoes select for transformation and interaction (−leu, −his +zeo); wherethe region labeled “A” represents yeast transformed with SrMV HC-Probait+SrMV HC-Pro prey; the region labeled “B” represents yeasttransformed with empty bait+RNase H-like protein prey; the regionlabeled “C” represents yeast transformed with lamin bait+RNase H-likeprotein prey; and the region labeled “D” represents yeast transformedwith SrMV HC-Pro bait+RNase H-like protein prey; although the figure isnot presented in color, the regions marked A and D on both plates appearblue, while the regions marked B and C on the −leu +zeo plate appearred;

FIG. 6A is the cDNA sequence of the RNase H-like protein (SEQ.ID.NO. 3)and FIG. 6B is the encoded amino acid sequence (SEQ.ID.NO. 4), accordingto an embodiment of the present invention;

FIG. 7A is the cDNA sequence of the sugarcane RING zinc finger proteinthat interacts with SrMV HC-Pro (SEQ.ID.NO. 5); FIG. 7B is the encodedamino acid sequence (SEQ.ID.NO. 6) according to an embodiment of thepresent invention;

FIG. 8A is the cDNA sequence of the LRR (leucine-rich repeat)transmembrane protein kinase that interacts with SrMV HC-Pro (SEQ.ID.NO.7); FIG. 8B is the encoded amino acid sequence (SEQ.ID.NO. 8) accordingto an embodiment of the present invention;

FIG. 9A is the cDNA sequence of a nucleic acid encoding the sugarcane14-3-3 protein that interacts with the RNase H-like protein (SEQ.ID.NO.9); FIG. 9B is the encoded amino acid (SEQ.ID.NO. 10), according to anembodiment of the invention;

FIG. 10A is the cDNA sequence of the sugarcane RING zinc finger proteinthat interacts with RNase H-like protein (SEQ.ID.NO. 11) and FIG. 10Bprovides the encoded amino acid sequence (SEQ.ID.NO. 12) according to anembodiment of the present invention;

FIG. 11A shows a 12% PA, CB stained gel; FIG. 11B shows a Large S-APprobed blot; in parts of FIG. 10, the molecular weight lane is indicatedas “MW”, lane 1 contains the E. coli expression product of Bugbuster™(Novagen, Madison, Wis., affiliate of Merck KgaA, Darmstadt, Germany)Insoluble pET30 with no insert; lane 2 contains the E. coli expressionproduct of Bugbuster™ Insoluble pET30 with an SrMV HC-Pro insert; lane 3contains one preparation of Ni column purified E. coli expressionproduct of Bugbuster™ Insoluble pET30 with an SrMV HC-Pro insert; andlane 4 contains a second preparation of Ni column purified E. coliexpression product of Bugbuster™ Insoluble pET30 with an SrMV HC-Proinsert;

FIG. 12 shows a two hour exposure of a 15% SDS PAGE gel containing RNaseH-like protein (lane labeled “RNase H”) labeled with ³⁵S Cysteineaccording to the present invention; control lanes are provided in whichno DNA was used in the preparation procedure (lane labeled “No DNA”) andin which Luciferase DNA was used (lane labeled “Luciferase”); molecularweight markers are indicated;

FIG. 13 shows a 32 hour exposure of a farwestern blot probed under invitro plant cell physiological conditions with a ³⁵S labeled RNaseH-like protein transcription and translation (TNT) product; the lanelabeled “His-S/HC-Pro” contains Ni column purified, His-S tagged SrMVHC-Pro protein produced in E. coli; the lane labeled “His-S” containsthe HIS-S tag only; the lane labeled “BSA” contains untagged bovineserum albumen; the lane labeled “HEWL” contains untagged hen egg whitelysozyme and the lane labeled “RNase A” contains untagged RNase A;

FIG. 14 shows a 6 hour exposure of a farwestern blot probed under invitro plant cell physiological conditions with a ³⁵S labeled RNaseH-like protein transcription and translation (TNT) product; the lanelabeled “His-S/HC-Pro” contains Ni column purified, His-S tagged SrMVHC-Pro protein produced in E. coli; the lane labeled “His-S/RNase H”contains Ni column purified, His-S tagged RNase H-like protein producedin E. coli; the lane labeled “His-S” contains the product in E. coli ofthe His-S tag only; the unlabeled lanes contain plant extracts which arefrom left to right, from: healthy sugarcane plant, SrMV infectedsugarcane plant, sugarcane plant transgenic for SrMV P1/HC-Pro,sugarcane plant transgenic for delta N 12 SrMV HC-Pro.; and the lanelabeled MWM contains molecular weight markers, which were used toproduce the size indicators on the side of the blot;

FIG. 15 shows a 32 hour exposure of a 15% SDS PAGE gel on which Nicolumn pull down products of a TNT ³⁵S labeled protein/His-S taggedprotein physiological incubation were run; the top lane marker indicatesthe source of DNA used in a TNT procedure in to obtain ³⁵S labeledprotein, where “RNase” indicates the use of RNase H-like protein cDNA,“No DNA” indicates a control in which no DNA template was provided, and“Luciferase” indicates a control in which Luciferase DNA was provided;the bottom lane marker indicates the His-S tagged product produced in E.coli, where “His-S/HC-Pro” or “HS/HC-Pro” indicates Ni column purifiedHis-S tagged SrMV HC-Pro protein, “His-S” indicates the His-S tag only,and “BSA” indicates untagged bovine serum albumen;

FIG. 16 shows a 3 hour exposure of a farwestern blot probed under invitro plant cell physiological conditions with a ³⁵S labeled RNaseH-like protein transcription and translation (TNT) product; the lanelabeled “His-S/14-3-3” contains Ni column purified, His-S taggedsugarcane 14-3-3 protein produced in E. coli; the lane labeled “His-S”contains the His-S tag only; the lane labeled “BSA” contains untaggedbovine serum albumen; the lane labeled “HEWL” contains untagged hen eggwhite lysozyme; and the lane labeled “RNase A” contains untagged RNaseA.

FIG. 17A depicts a DNA construct containing sense and antisensesequences in opposite directions and separated by an intron which,following transcription to mRNA, form a double strand due tosense/antisense sequence complementation shown in FIG. 17B;

FIG. 18 depicts the pMCG161 plasmid expression vector, according to oneembodiment of the present invention;

FIG. 19 depicts the results of transient expression of constructsinserted in the pMGC161 plasmid in embryonic sugarcane calli where thecallus 1 is transformed with GUS under control of a Ubi promoter; callus2 is transformed with GUS under control of a Ubi promoter and DNA thatproduces double stranded GUS mRNA under control of a 35Sint promoter;callus 3 is transformed with the same constructs as callus 2 andadditionally with DNA that produces double stranded RNase H-like proteinmRNA under control of the 35Sint promoter; callus 4 is transformed withthe same constructs as callus 2 and additionally with SrMV P1/HC-Prounder the control of the Ubi promoter; callus 5 is transformed with thesame constructs as callus 2 and additionally with SrMV HC-Pro-delta N 12under control of the Ubi promoter for ease of plasmid construction toinclude a start ATG, the first N-terminal amino acids of the full lengthSrMV HC-Pro are deleted via deletion of 36 cDNA base pairs); and callus6 is transformed with the same constructs as callus 2 and additionallywith DNA that produces double stranded GFP under control of the 35Sintpromoter; although the figure is not provided in color, all darkerregions in the calli of the figure are blue while lighter regions arepinkish tan, as will be apparent to one skilled in the art;

FIG. 20 is a graphical representation of three experiments such as thatof FIG. 19; vertical bars represent the average of these threeindependent experiments while vertical line represent standard errors;treatment 1 represents transformation with GUS under control of a Ubipromoter; treatment 2 represents transformation with GUS under controlof a Ubi promoter and DNA that produces double stranded GUS mRNA undercontrol of a 35Sint promoter; treatment 3 represents transformation withthe same constructs as treatment 2 and additionally with DNA thatproduces double stranded RNase H-like protein mRNA under control of the35Sint promoter; treatment 4 represents transformation with the sameconstructs as treatment 2 and additionally with RNase H-like proteinunder control of the 35Sint promoter; treatment 5 representstransformation with the same constructs as treatment 2 and additionallywith SrMV P1 and HC-Pro under the control of the Ubi promoter; treatment6 represents transformation with the same constructs as callus 2 andadditionally with SrMV HC-Pro-delta N 12 under control of the Ubipromoter; and treatment 7 represents transformation with the sameconstructs as callus 2 and additionally with DNA that produces doublestranded GFP MRNA under control of the 35Sint promoter;

FIG. 21 depicts 4 embryonic calli transiently transformed withconstructs including, in callus 1, GUS under control of the 35Sintpromoter; in callus 2, GUS under control of the 35Sint promoter and DNAthat produces double stranded GUS mRNA also under control of the 35Sintpromoter; in callus 3, the same constructs as callus 2 and additionallyRNase H-like protein under control of the 35Sint promoter; in callus 4,the same constructs as callus 2 and additionally DNA that produces RNaseH-like protein mRNA under control of the 35Sint promoter; although thefigures is not presented in color, the darker colored calli, calli 1 and4 are blue while the lighter colored calli, calli 2 and 3 are pinkishtan was will apparent to one skilled in the art.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS OF THE INVENTION

The present invention includes a novel method for determination of plantproteins active in PTGS or suppressive of PTGS. The invention may beused in both monocot and dicot plants, including sugarcane.

The methods of the present invention may be used, inter alia: (i) toidentify, isolate and characterize cellular proteins that interact withthese PTGS suppressive viral proteins or plant proteins involved in PTGSusing a yeast two-hybrid system; and (ii) to evaluate these plant/viralprotein interactions in vitro under physiological conditions and in vivoin either transient studies or in transgenic plants, such as sugarcane,expressing these viral proteins. Because many viral proteins such as P1and HC-Pro are multifunctional proteins involved in various aspects ofplant/potyvirus interactions, the methods may also be used to assess therole of interacting host proteins in gene silencing. The methods may becarried out using a combination of molecular genetics, immunologicalstudies, transient antisense suppression studies, and planttransformation.

The overall method includes using a yeast two-hybrid system to searchfor plant proteins that interact with viral suppressors of PTGS toidentify proteins involved in PTGS. These proteins or proteinsidentified as having a role in PTGS through other methods may then serveas bait to locate other proteins involved in PTGS. Because proteins thatinteract with other proteins involved in PTGS may be either suppressiveof or active in PTGS, the yeast two-hybird assays may identify bothtypes of proteins. Whether the proteins involved in PTGS are active inor suppressive of PTGS may be determined through a variety of methods,including identification of motifs with particular functions, comparisonwith known proteins, and in planta studies.

In the present invention the bait protein may be either active in PTGSor may function as a suppressor. The prey used is derived from anexpression library of the plant of interest. In an exemplary embodimentof the invention, the prey library is derived from a plant in which PTGSis occurring. This facilitates identification of proteins involved inPTGS because such proteins are actively being produced in the silencedplant and may therefore be better represented in the mRNA pool of asilenced plant than in a non-silenced plant.

After identification of an interacting prey, the bait and prey portionsof the two-hybrid screen of the present invention may be reversed tohelp identify false positives. Comparison with controls designed to lookfor activation of the reporter system absent either the bait or prey mayalso be used to identify false positives.

Such yeast two-hybrid screens may then be followed by assays such afarwestern blots or pull down assays to further determine whetheridentified proteins are false positives and also to characterize theirfunction. In planta physiological conditions may be used in such assays.

In planta studies may also be conducted using transiently or permanentlytransformed embryonic calli transformed with either a test protein or aDNA encoding a double stranded mRNA of the test protein (which inducesPTGS of that protein) in order to further evaluate the suppressive oreffective role of the test protein in PTGS.

Proteins identified in one or more of the above screens may also befurther characterized by identifying putative functional domains andalso be searching for overall cDNA and amino acid sequence similaritywith other proteins.

Often proteins and cDNAs identified using the above methodologies may benovel and patentable. In the present invention, five such novel proteinsand cDNAs have been identified: the sugarcane 14-3-3 protein and cDNA,the RNase H-like protein and cDNA, the sugarcane LRR transmembraneprotein kinase and cDNA, and the two sugarcane RING zinc finger proteinsand cDNAs. Additionally, the SrMV P1/HC-Pro protein and cDNA used insome embodiments of the method of the present invention are also novel.

The above cDNAs as well as other identified using the methodologies ofthe present invention may also be used to construct transgenic plants,plant cells and plant tissues (collectively “plant entities”) in whichPTGS is either enhanced or suppressed. The methodologies used in theassay methods to generate embryonic calli and other methods know to theart may be used to construct these transgenic plant entities.Transformation may be transient or permanent, depending upon theintended use of the transgenic plant entity.

Permanently transformed plant entities with increased PTGS may be virusresistant. PTGS may be suppressed in other plants to allow increasedexpression of a transgene for any of a variety of reasons, includingimprovement of plant health, adaptation to certain growing conditions,producing of a novel nutrient or vaccine, and production of a proteinlater purified for medical or industrial uses. Finally, thePTGS-regulatory methods of the present invention may be used to inducePTGS of a particular gene, for instance by introducing a constructencoding dsRNA for a portion of the gene, thereby offering a novelmethod of producing knock-out plants.

The following examples are provided only to illustrate certain aspectsof the invention and are not intended to embody the total scope of theinvention or any aspect thereof. Variations of the exemplary embodimentsof the invention below will be apparent to one skilled in the art andare intended to be included within the scope of the invention.

EXAMPLES Example 1 Nucleotide Sequence of Sorghum Mosaic Virus

SrMV is a member of the genus Potyvirus and can cause mosaic disease andyield loss in poaceous plants such as sugarcane and sorghum (Shukla etal., 1994). A 2.0-kb region located at the 3′ end of the SrMV strain Hgenomic RNA which encompasses the 3′ untranslated region, the completeopen reading frame for the coat protein and part of the NIb ORF has beenpreviously sequenced (Yang and Mirkov, 1997). The remaining part of theSrMV genomic RNA has been sequenced in the present invention fromoverlapping RT-PCR products. From the combined sequences, a 9,581 bpconsensus was derived that contains a continuous ORF encoding a 3079amino acid putative polyprotein with ten gene products typical formembers of the genus Potyvirus (Ingelbrecht et al., in preparation). TheSrMV polyprotein consensus sequence was compared to that of Tobacco etchvirus (TEV-HAT), Maize dwarf mosaic virus (MDMV-Bu), Plum pox virus(PPV-D) and Pea seedborne mosaic virus (PSbMV-D) in a multiple sequencealignment using Clustal X. Based on this alignment, putative proteolyticcleavage sites were positioned in the SrMV polyprotein. (See FIG. 1.) Anovel nucleic acid (SEQ.ID.NO. 1) encoding a novel SrMV P1/HC-Proprotein (SEQ.ID.NO. 2) was developed.

Example 2 SrMV P1/HC-Pro Reverses PTGS

The potyviral HC-Pro is a multifunctional protein and harbors at least 3functional domains. The N-terminal part contains a highly conserved KITCmotif and is involved in aphid transmission (Thornbury et al., 1985;Atreya and Pirone, 1991). The central part is required for long distancemovement and virus amplification (Cronin et al., 1995; Kasschau et al.,1997) while the C-terminal part represents a papain-like proteinaseinvolved in polyprotein processing (Carrington et al., 1990). Inaddition, potyviral HC-Pro is the determinant of potyviral synergisticinteractions with other viruses (Pruss et al., 1997).

Potyviral HC-Pro can reverse the effects of PTGS in tissues that werepreviously silenced. This suppression phenotype has been observed forthe potyviral HC-Pro protein of three different potyviruses, TEV, PVYand PSbMV, suggesting that this particular function of potyviral HC-Promay be conserved among different potyviruses (Voinnet et al., 1999).From this combined with the presence of conserved amino-acid sequencemotifs in the SrMV HC-Pro, it was postulated that the SrMV HC-Pro mightalso act as a suppressor of PTGS in sugarcane. However, the suppressorability of SrMV HC-Pro could not be assumed based on sequence similarityalone and experiments were required to determine if suppressor activitywas exhibited in sugarcane and the extent of such suppression.

Reversal of PTGS by SrMV P1/HC-Pro was demonstrated by retransforming aplant which is posttranscriptionally silenced for the CP transgene(Ingelbrecht et al., 1999, plant #16). The silenced plant #16 hadoriginally been generated by selection on bialaphos. Embryogenic callusgenerated from plant # 16 was therefore retransformed with either nptIIalone or with nptII and SrMV P1/HC-Pro, and transgenic plants wereselected on geneticin. As shown in the Northern blot analysis of the CPgene in FIG. 2, in 5 of the 6 plants retransformed with nptII and SrMVP1/HC-Pro, PTGS has been suppressed and the steady state levels of theCP mRNA are equivalent to the levels seen in a plant that is notsilenced (in FIG. 2, NS=plant #463 of Ingelbrecht et al., 1999). No suchreversal was seen in plants retransformed with nptII alone. Southern andNorthern analysis of nptII and SrMV P1/HC-Pro on these plants revealedthat the one plant retransformed with nptII and SrMV P1/HC-Pro in whichPTGS was not reversed was only transgenic for nptII (data not shown).

Example 3 The Yeast Two-Hybrid System

The yeast two-hybrid system has become a powerful tool for the study ofprotein-protein interactions in vivo. It can also be used to search anexpression library for proteins that interact with a target protein.This latter application is being increasingly used with success in bothanimal (Kleiman and Manley, 1999) and plant systems (Kohalmi et al.,1998; Gindullis et al., 1999). Currently, high throughput two-hybridprocedures are being developed to catalogue protein-protein interactionson a genome-wide scale (Walhout et al., 2000).

Before embarking on a screen, it is useful to study the behavior of thetarget protein, termed bait, in the yeast cells. For example, one shoulddemonstrate that the bait protein by itself does not activatetranscription of the marker genes that are used in the selection andscreening procedures. False positives can still be encountered followingthis precaution, but the great majority of these can be eliminated insubsequent steps. Finally, protein-protein interactions identified inthe yeast cells may be verified via independent experimental procedures,preferably in the original biological system or a physiologicallyequivalent environment.

A yeast two-hybrid screening procedure typically consists of thefollowing steps:

1) Subclone the coding region of the target protein into a LexA or GAL4binding domain vector to create a bait. In an example of the presentmethod this bait is the SrMV HC-Pro sequence cloned into pHYBLexZeo togenerate pIVING1281 or SrMV P1 cloned into pAS2 to generatepIVING1088-6.

2) Transform the bait into the yeast reporter strain to verify that theexpressed protein does not activate the reporter gene(s) by itself andis not toxic to the yeast cells. In an example of the present method theyeast strain L40 is transformed with either pIVING1168, pIVING1281, orpIVING1088-6 alone, and also co-transformed with either a pGAD424derivative (pIVING1154) that lacks an insert, empty pHYBLexZeo, orpHYBLexZeo containing a non-specific bait (lamin). These sets oftransformed yeast strains function as negative controls for thescreening procedures as known in the art.

3) Subclone a cDNA library of sequences into a GAL4 activator domainvector to create a library of prey. In an example of the present system,the cDNA library is derived from a transgenic sugarcane plant thatdisplays PTGS (Ingelbrecht et al., 1999, plant #16) and is cloned into amodified pGAD424 derivative (pIVING1154), which constitutes the prey.

4) Transform the yeast reporter strain carrying the bait plasmid withthe cDNA/prey library.

5) Screen yeast bait and prey co-transformed colonies for expression ofa reporter gene that is fused to a GAL4 activated promoter. In anexample of the present system, the yeast strain L40 contains reporterconstructs for expression of a β-galactosidase gene, as well as areporter construct for the HIS gene. Cells in which bait and prey fusionproteins interact in the yeast nucleus will grow on minimal media in theabsence of histidine and will produce β-galactosidase enzyme activity atlevels elevated from the negative control cells .

6) Verify positive interactions of co-transformants and eliminate falsepositives by reestablishing and retesting yeast strains, testing yeaststrains which only contain the previously identified prey construct andtesting the prey construct with empty bait or bait that contains anon-specific protein (such as lamin). If the prey construct activatestranscription of a reporter gene that is under regulation of aGAL4-inducible promoter under any of the above conditions, the prey maybe considered a false positive.

This method, as used in the examples of the present system, may be usedto isolate host proteins from sugarcane and other plants that areinvolved in PTGS using the HC-Pro and P1 proteins from SrMV or otherproteins involved in PTGS as bait. Experiments using a library-scaleyeast two-hybrid screen with SrMV HC-Pro as bait have identified 441interacting proteins. At least 12 of these (including a protein withhigh similarity to a viral RNase H referred to herein as the “RNaseH-like protein”) have been confirmed to be true interactors. Additionalproteins involved in PTGS, such as a novel sugarcane 14-3-3 protein,have also been identified using the above method based on their abilityto interact with the RNase H-like protein used as bait.

Example 4 Self-Interaction with HC-Pro or P1 in a Yeast Two-HybridSystem

The yeast two-hybrid method of this invention may be used to identifyproteins from sugarcane that interact with HC-Pro or P1 of SrMV. Thetwo-hybrid procedure is based on the reconstruction of a functionaltranscriptional activator such as GAL4 or LexA, whose DNA binding domain(DBD) and transcription activation domain (AD) are expressed on twodifferent vectors (Fields and Song, 1989). In the present example, thebait protein, i.e. the SrMV HC-Pro, was fused to the DNA-binding domainand the cDNA library was constructed in the activation domain vector,which produces the prey. These vectors were introduced into the yeaststrain L40, which has an endogenous β-galactosidase (lacZ) reporter geneand the nutritional marker HIS3 for selection.

A 1.4-kb RT-PCR fragment encompassing the complete open reading frame ofHC-Pro was amplified from SrMV virion particles and subcloned inpCR4-TOPO by T/A cloning (Invitrogen, Carlsbad, Calif.) yieldingpIVING1148-1. The complete HC-Pro ORF was cloned as a fusion proteininto the two-hybrid vectors pHYBLexZeo (bait) and pGAD424 (prey),yielding the plasmids pIVING1281 and pIVING1168, respectively. The P1ORF was subcloned as a 0.78-kb fragment in pAS2 yielding pIVING1088-6.The vector-insert junctions were confirmed by sequencing for allconstructs.

Recent studies have shown that potyviral HC-Pro proteins can interactwith themselves in a yeast two-hybrid system (Urcuqui-Inchima et al.,1999; Guo et al., 1999), in agreement with an earlier proposal that thepotyviral HC-Pro is biologically active as a homodimer (Thornbury etal., 1985). The ability of the SrMV HC-Pro for self-interaction wastested in the present example. It was also verified that the SrMV HC-Probait construct does not activate transcription of the lacZ gene, eitherby itself or in combination with empty bait or prey vectors. Theselatter experiments were also conducted for the P1 bait constructpIVING1088-6. As shown in FIG. 3, the SrMV HC-Pro does interact withitself (A) in the two-hybrid system demonstrating the functionality ofthis construct. Secondly, no lacZ expression can be observed incombination with either empty prey (BY or empty bait (D) vectors or abait vector containing a non-specific protein (lamin; C). Therefore,SrMV HC-Pro does not activate transcription by itself and can be used asbait to screen for interacting proteins. Similar results were obtainedfor the SrMV P1 bait construct (data not shown).

Example 5 Development of a cDNA Library from Silenced Sugarcane

The development of SrMV-resistant sugarcane plants via transformationwith an untranslatable form of the capsid protein sequence has beenpreviously described and it has been demonstrated that the underlyingresistance mechanism is related to PTGS (Ingelbrecht et al., 1999). Asdescribed in Ingelbrecht et al., 1999, plant #16 is a recovery plantthat is immune to infection with SrMV after recovery from the initialinfection and is posttranscriptionally silenced for the CP transgene.Although the CP transgenes are actively transcribed, the CP steady-statemRNA level is below the detection limit on an RNA gel blot (See Example2 and FIG. 2).

Using poly(A)+ RNA from silenced leaf tissue of this plant a cDNA fusionlibrary was constructed in the prey vector PIVING 1154 using the SMART™PCR cDNA library construction kit (offered by Clontech, Palo AltoCalif., a part of Beckton Dickinson, Franklin Lakes, N.J.). To createthe vector plVING1154, pGAD424 was modified to allow directional cloningof the cDNA into asymmetric SfiI sites. The library has an estimated1.6×10⁶ primary clones. As shown in FIG. 4, the inserts range between0.5 to 2.0 kb with an average size of about 0.8 kb. Also, a 0.8-kbfragment containing the CP coding sequence could be readily amplifiedfrom the plasmid cDNA library indicating that low abundance mRNAs arerepresented.

Example 6 Library Scale Two-Hybrid Screen with HC-Pro Bait

The yeast strain L40 containing the SrMV HC-Pro bait construct wastransformed with the prey CDNA library representing the equivalent ofapproximately 1.6×10⁶ primary E. coli clones (see Example 4). About 77million primary yeast transformants were plated on selective media(−leucine, −histidine +zeocin) and 776 yeast colonies were recovered bythe fourth day of growth. Of these, 441 showed a range of light to heavyblue staining in the standard β-galactosidase colony-lift filter assay.To date, all of these double transformants have been colony purified,and all of the prey plasmids have been isolated. A RNase H-like protein,a RING zinc finger protein and LRR transmembrane protein kinase havebeen identified among the true positives. Some results of this screenand additional screens with SrMV P1 or RNase H like-protein as the baitare depicted in Table 1.

TABLE 1 Bait (DBD) Prey (AD) Interaction SrMV HC-Pro SrMV HC-Pro ++ SrMVP1 SrMV P1 −/+ SrMV HC-Pro SrMV P1 − SrMV P1 SrMV HC-Pro − SrMV HC-ProRNase H-like prot. +++ RNase H-like prot. SrMV HC-Pro +++ SrMV P1 RNaseH-like prot. − RNase H-like prot. RNase H-like prot. +++

A −/+ plus designates that very light blue staining was observed onlyafter a 24 hour incubation. +++ designates that strong blue staining wasseen in less than 30 minutes, and ++ designates strong blue staining inless than 3 hours. The RNase H-like protein self interaction mayindicate that the protein functions as a homodimer.

Example 7 RNase H-Like Protein

One of the prey plasmids that was recovered during the experiments ofExample 5 was retransformed into the SrMV HC-Pro bait yeast strain toreconfirm activation of the reporter genes. The plasmid encodes aprotein with approximately 40% identity to a viral RNase H. This proteinhas been recovered several times using the methods of these examples. Asshown in FIG. 5, the interaction with HC-Pro is very strong (D) andthere is no interaction in combination with an empty bait plasmid orwith a lamin bait plasmid (B and C, respectively). Furthermore, theRNase H-like gene is in the correct reading frame with respect to thefusion protein, so this is a true interactor. The cDNA sequence(SEQ.ID.NO. 3) for a nucleic acid encoding the RNase H-like protein andits amino acid sequence (SEQ.ID.NO. 4) are provided in FIG. 6.

Example 8 RING Zinc Finger Protein that Interacts with HC-Pro

Another protein identified as a true positive in HC-Pro interactionscreens is a RING zinc finger protein. A partial cDNA sequence of anucleic acid encoding this protein is provided in FIG. 7A (SEQ.ID.NO.5). The encoded amino acid sequence is included as FIG. 7B (SEQ.ID.NO.6). The nucleic acid sequence is sufficient to identify nucleic acidsencoding the protein, but likely lacks approximately 300 bp at the 5′end in the figure. However, because a protein encoded by the possiblytruncated nucleic acid sequence was identified in a two-hybrid screen, anucleic acid with the sequence provided must encode a sufficient portionof the protein to allow interaction with SrMV HC-Pro. With theinformation disclosed in this invention, it is possible for a personskilled in the art to isolate the full length cDNA and protein sequence.

Example 9 LRR Transmembrane Protein Kinase

Another protein identified as a true positive in SrMV HC-Pro yeasttwo-hybrid screens is an LRR transmembrane protein kinase. A partialcDNA sequence of a nucleic acid encoding this protein is provided inFIG. 8A (SEQ.ID.NO. 7). The encoded amino acid sequence is included inFIG. 8B (SEQ.ID.NO. 8). The sequence is sufficient to identify nucleicacids encoding the protein, but likely lacks approximately 1 kb at the5′ end in the figure. However, because a protein encoded by the possiblytruncated nucleic acid sequence was identified in a two-hybrid screen, anucleic acid with the sequence provided must encode a sufficient portionof the protein to allow interaction with SrMV HC-Pro. With theinformation disclosed in this invention, it is possible for a personskilled in the art to isolate the full length cDNA and protein sequence.

Example 10 Library Scale Two-Hybrid Screen with RNase H-Like Protein asBait

A library scale two-hybrid screen similar to that of Example 5 wasconduced using the same prey library (described in Example 4) with theRNase H-like protein as bait. Using the RNase H-like protein as bait, atleast two true positives for prey/host proteins were identified andfurther characterized. The results of some of these assays as well asassays with SrMV HC-Pro and SrMV P1 as bait are depicted in Table 2.

TABLE 2 Bait (DBD) Prey (AD) Interaction SrMV HC-Pro SrMV HC-Pro ++RNase H-like prot. SrMV HC-Pro +++ SrMV P1 RNase H-like prot. − RNaseH-like prot. RNase H-like prot. +++ RNase H-like prot. Sugarcane 14-3-3+++ SrMV HC-Pro Sugarcane 14-3-3 − SrMV P1 Sugarcane 14-3-3 −

A −/+ designates that very light blue staining was observed only after a24 hour incubation. +++ designates that strong blue staining was seen inless than 30 minutes, and ++ designates strong blue staining in lessthan 3 hours.

Example 11 Sugarcane 14-3-3 Protein

One protein identified through its ability to interact with the RNaseH-like protein is a sugarcane 14-3-3 protein. The sequence of a nucleicacid encoding the sugarcane 14-3-3 protein is provided in FIG. 9A (SEQ.ID. NO. 9). The encoded amino acid sequence is provided in FIG. 9B (SEQ.ID. NO. 10). The role of another 14-3-3 protein in signal transductionin the dicot Arabidopsis has recently been described in Sehnke et al.,2002. A similar role in monocots is suggested by the disclosed in thisinvention. Specifically, 14-3-3 proteins exhibit aphosphorylation-dependent association with proteins in dicots. The RNaseH-like protein of the present invention contains a serine with a 99.1%chance of being phosphorylated. This serine is also located in a goodconsensus 14-3-3 protein client-binding site. More specifically, theputative phosphorylation/binding site is VIQNpSPPDL (wherein “pS”designates phosphoserine) (SEQ. ID. NO. 13) beginning at amino acid 48of the protein in FIG. 6B. Binding of sugarcane 14-3-3 and the RNaseH-like protein in order to achieve a functional dimer (or as part of afunctional complex containing other proteins) is also suggested by theabsence of DNase or RNase activity of either protein in isolation. Asshown in Table 2, 14-3-3 does not interact noticeably with SrMV HC-Proor SrMV P1.

Example 12 RING Zinc Finger Protein that Interacts with RNase H-LikeProtein

Another protein identified as a true positive in RNase H-like proteininteraction screens is a RING zinc finger protein. This is not the sameRING zinc finger protein identified as interacting with SrMV HC-Pro. Apartial cDNA sequence of a nucleic acid encoding this protein isprovided in FIG. 10A (SEQ.ID.NO. 11). The amino acid sequence encoded bythe nucleic acid is provided in FIG. 10B (SEQ.ID.NO. 12)

Example 13 DNA Sequence Analyses of the Prey Genes Identified in theTwo-Hybrid Screen

Prey sequences that have been verified by the above screening proceduresmay be DNA sequenced utilizing standard fluorescence-based thermocyclesequencing methods, and restriction maps created. This sequenceinformation may be utilized to search the genetic databases with BLASTxand BLASTp to determine sequence similarity with known genes orproteins. In cases where similarity is clear, one may verify that thesequence under investigation is inserted in the appropriate readingframe in the prey vector. If the sequence is not in the appropriatereading frame, the prey can be considered a false positive.

Example 14 Characterization of the Prey Nucleic Acids Identified in theTwo-Hybrid Screen for the Ability of their Corresponding Proteins toInteract with SrMV HC-Pro Protein or other Bait Protein in Vitro UnderPhysiological Conditions

Although prey and bait combinations identified in the two-hybrid screenof the present invention may represent proteins which interact in yeastcells, several caveats of the assay may produce results which are notindicative of interactions that occur in planta. The yeast two-hybridsystem assay requires that the bait and prey GAL4 fusion proteins bothbe imported into the yeast nucleus, which biochemically is a much morereducing environment than the yeast cytoplasm. This may lead todifferent patterns of protein folding than might otherwise occur forproteins whose operating environment is a more oxidative cytoplasm.Additionally, protein fragments placed in an unnatural position in ayeast two-hybird assay (e.g. an N term region in the C term of thefusion protein) may fail to fold or function properly because ofpositional effects.

Given that in vivo studies often require specialized reagents in thepresent method, an initial in vitro screening procedure in whichpotential protein interactions are verified under physiologicalconditions may be used to save time and expense associated with planttransformations in the case of false positives. However, it does remainpossible that in vitro conditions may lack certain requirements forinteraction found only in living cells and thus generate falsenegatives. Therefore, in some instances it may be desirable to proceedwith in planta studies without or despite results in vitro. Such anapproach is within the scope of the present invention.

If in vitro studies are preformed, host/prey proteins identified in thetwo-hybrid screen may be further tested for their ability to interactwith SrMV P1 and SrMV HC-Pro or other proteins involved in PTGS in vitrounder physiological conditions (i.e. pH 6.8 and 0.1 M ionic strength) aseither polyhistidine, S-tag, or glutathione-s-transferase (GST)fusion-proteins. A series of vectors from Novagen, Inc. (Madison, Wis.,affiliate of Merck KgaA, Darmstadt, Germany) including pET15b,pET29a,b,c and pET30a,b,c, allow the construction of N-terminal orC-terminal polyhistidine fusion proteins, which in the case of pET29 andpET30 derivatives may also contain S-tags. Pharmacia Corp. (Peapack,N.J.) also produces pGEX2, pGEX3 and pGEX4 derivatives in which GSTfusion proteins may be produced. These vectors allow the specificpurification of “tagged” proteins that have either polyhistidine tags(using Ni+-chelated resin), S-tags (using S-agarose), or GST-tags (usingreduced glutathione-conjugated resins). Bait and prey inserts identifiedfrom the two-hybrid screen may be subcloned into one of these vectors.Other vectors encoding tag regions may also be used.

One approach is to construct two vector types, if possible, for the baitas well as for each prey that has been identified and to produce GST-and polyhistidine tagged proteins in E. coli. These tagged proteins maybe purified with kits available from Pharmacia, Novagen, or othersources, or using methods known to the art and appropriate for theselected tag. His-S tagged SrMV HC-Pro produced in the pET30 vectorpurified with a Ni column is shown in FIG. 11 in lanes 3 and 4. As FIG.11 indicates, tagged protein is produced only when an insert is presentin the vector and may be largely purified using a Ni column.

Radiolabelled SrMV HC-Pro, SrMV P1, RNase H-like protein or other baitproteins of interest may be produced by translation of theircorresponding transcripts in rabbit reticulocyte lysate. Using the TNSmethod, transcripts may be generated in a T7 in vitro transcriptionsystem or other system known to the art. Subsequent translation in thepresence of S-35-labeled methionine or cysteine allows radiolabeling ofthe bait protein. FIG. 12 shows that RNase H-like protein produced usingthe above methodology is, in fact, radiolabeled. A small amount of thistranslated lysate may then be incubated with the tagged prey proteinbound to its corresponding resin, and retention of radiolabeled baitprotein may be assayed with SDS-PAGE gels and autoradiograms. Positivecontrols can include tagged bait protein, and negative controls caninclude the tags themselves, as well as the resins without any otherproteins. These procedures or various modifications thereof readilydetermined by one skilled in the art allow one to determine whetherprospective prey proteins are capable of interacting with either one orboth viral proteins in vitro under physiological conditions.

Example 15 Farwestern Blots and Other Assays Indicate Interaction ofVarious Proteins

FIG. 13 shows a farwestern blot of His-S tagged SrMV HC-Pro probed with³⁵S labeled RNase H-like protein. Both proteins were produced accordingto the methods of the above examples. A comparison of binding of labeledRNase H-like protein to the His-S tagged SrMV HC-Pro to the variouscontrols indicates that the interaction is specific between the twoproteins and also that it occurs in conditions approximating plantphysiological conditions.

FIG. 14 shows a farwestern blot of His-S tagged SrMV HC-Pro and RNaseH-like protein and a plant extracts probed with ³⁵S labeled RNase H-likeprotein. A comparison of the lanes indicates that the RNase H interactswith itself and SrMV HCPro and with other sugarcane proteins—from leftto right—healthy sugarcane plant, SrMV infected sugarcane plant,sugarcane plant transgenic for SrMV P1/HC-Pro, sugarcane planttransgenic for SrMV delta N 12 HC-Pro. An extra band in the three plantsexpressing SrMV HC-Pro that is slightly smaller than the tagged SrMVHC-Pro may be seen in FIG. 14.

FIG. 15 shows the results of a Ni column pull down of His-S taggedprotein and its binding partner. The proteins attached to the Ni columnwere removed and run on an SDS PAGE gel. The results indicate that His-Stagged SrMV HC-Pro and not control proteins pulled down labeled RNaseH-like protein. Label controls were not pulled down. This indicates thatthe SrMV HC-Pro/RNase H-like protein interaction is specific and viableunder physiological conditions.

FIG. 16 shows a farwestern blot of His-S tagged sugarcane 14-3-3 probedwith ³⁵S labeled RNase H-like protein. A comparison of binding oflabeled RNase H-like protein to the His-S tagged sugarcane 14-3-3 to thevarious controls indicates that the interaction is specific between thetwo proteins and also that it occurs in conditions approximatingphysiological conditions.

Example 16 HC-Pro Deletion Mutant and Interaction with RNase H-LikeProtein

In order to better determine the region of SrMV HC-Pro responsible forinteraction with the RNase H-like protein, a series of deletion mutantswere created and their interaction was tested in yeast two-hybrid andfarwestern blot assays as described above. The results of theseexperiments are summarized in Table 3 and indicate that the C terminalportion, including the last 10 kDa of the HC-Pro protein are requiredfor interaction with the RNase H-like protein while the N terminalregion up to 7 kDa is not necessary.

TABLE 3 HC-Pro segment Assay Interaction Full length Yeast two-hybrid+++ ←--------------→ Full length Farwestern +++ ←--------------→ N 1.3kDa deletion Yeast two-hybrid +++ ←-------------→ N 7.0 kDa deletionYeast two-hybrid +++ ←---------→ C 10 kDa deletion Farwestern −←---------→ C 20 kDa deletion Farwestern − ←----→ +++ in yeasttwo-hybrid assays indicates that strong blue staining was seen in lessthan 30 minutes. +++ in farwestern assays indicates that binding wasapparent after a short exposure time.

Example 17 Complete Cloning of Partial cDNA Prey Clones

Candidate clones whose corresponding protein sequences interact withSrMV HC-Pro or other bait proteins in vitro under physiologicalconditions may be completely cloned with 5′ RACE procedures or otherprocedures known to the art.

Example 18 Production of Antiserum

To facilitate in vivo co-immunoprecipitation studies further outlinedbelow, antisera recognizing SrMV HC-Pro or another protein involved inPTGS may be produced. Using a pET or pGEX expression plasmid constructas described above or another 6xhis construct known to the art, one mayproduce 6Xhis-tagged SrMV HC-Pro or other protein in E. coli, purify thefusion protein as described above, and utilize the protein as an antigenwith rabbits or other animals to produce polyclonal antisera. Similarly,SrMV P1 and host proteins may also be utilized to produce antisera,depending upon the potential utility of the resultant serum. Otherantisera production techniques may also be used to produce polyclonalor, where useful, monoclonal antibodies.

Example 19 Transgenic Plant Studies

Because of its specific mode of action, the SrMV P1/HC-Pro and otherproteins identified as involved in PTGS using the above two-hybrid andin vitro methods may target one or more factors that are expressed andfunctional in silenced tissue. This may be confirmed in planta. Becausethe cDNA library in the above examples was constructed from a plantharboring a PTGS-silenced SrMV coat protein sequence, and the plant isresistant to SrMV, one cannot readily utilize mechanical transmission ofSrMV as a source of SrMV HC-Pro or other viral proteins in such plants,and thus may resort to transgenic methodologies.

Plant transformation studies demonstrate the feasibility of thisapproach. To date, SrMV HC-Pro transformants and SrMV P1/HC-Protransformants have been confirmed via Southern and Northern analyses.The plants were produced via particle gun transformation of embryogeniccallus, as previously described (Ingelbrecht et al., 1999). Threedifferent plant transformation vectors have been constructed: (1)pIVING1023 which contains the SrMV P1/HC-Pro polyprotein sequence, inwhich SrMV HC-Pro may be expected to be cleaved out by the SrMV P1protease in planta, (2) pIVING1002 which contains only the SrMV P1sequence, and (3) pIVING991-1 which contains only the SrMV HC-Prosequence. In the present example, two selectable markers for sugarcanetransformation have been used, the nptII gene in combination withgeneticin, and the bar gene for selection with bialaphos. Bialaphosselection may be used on bar transformed cells, and geneticin selectionmay be used on bar transgenic cells to select for second transformationevents of previously transformed tissue. Other transformation mechanismsand selection systems known to the art may also be used. Suchsequentially transformed plants may serve as a source of material for inplanta studies.

In a further example, the transgenic plant #16 that was used as a sourcefor construction of the prey cDNA library above, was utilized togenerate embryogenic callus. Plant #16 was previously transformed withthe bar gene, and in this example was transformed again with the SrMVP1-HC-Pro construct using geneticin selection. Because SrMV P1 has beendescribed as an enhancer of the PTGS-inhibition by SrMV HC-Pro, thecapsid message levels may be higher in SrMV P1-HC-Pro transformed #16plants as compared to SrMV HC-Pro transformed #16 plants. In addition tohaving a system of modulated PTGS-suppression, these transgenic plantsalso allow verification in planta of the interactions between the viralproteins and the identified prey proteins, utilizing SrMV HC-Prospecific antiserum in standard co-immunoprecipitation methodologies.

Sequence homology of the isolated host proteins with known proteins, ifpresent, may be used to postulate their biological function and whetheror not they are involved in PTGS in a manner similar to that employedwith the genes identified in mutagenesis studies of Neurospora, C.Elegans and Arabadopsis.

One approach for determining functions of the isolated genes may bebased on creating suppression phenotypes for these genes in sugarcanevia genetic transformation. Efficient triggering of gene suppression canbe achieved by simultaneous expression of sense and antisense constructsor constructs designed to produce dsRNA as demonstrated in tobacco, riceand Arabadopsis (Waterhouse et al., 1998; Smith et al., 2000; Chuang andMeyerowitz, 2000). See also FIG. 17 for depictions of the basicstructure of DNA and resulting mRNA for such constructs. Such constructsmay be created for each of the isolated host genes and introduced inplants that display PTGS of a capsid protein transgene, such as plant#16. The transformation methodologies described above can be followed togenerate stably transformed plants. Alternatively, a protoplast-basedsystem can be used in combination with electroporation or PEG-mediatedtransformation. Expression of the capsid protein will be analyzed andreversal of silencing may indicate that the PTGS mechanism in thesetransgenics is impaired and that the transformed host genes are involvedin PTGS.

Example 20 In Planta Studies of HC-Pro and RNase H-Like Protein

Embryonic sugarcane calli were bombarded with various constructsaccording to the above examples in order to provide transient expressionto verify the role of HN-Pro and RNase H-like protein in PTGS. Doublestranded mRNA relating to the various proteins was generated usingconstructs such as those show in FIG. 17 inserted into the vector ofFIG. 18.

FIG. 19 shows that GUS under a Ubi promoter alone is expressed inembryonic callus. Introduction of a double stranded GUS mRNA reduces itsexpression by activating PTGS of GUS mRNA. However expression isrestored by both introducing SrMV P1-HC-Pro into the callus and byinducing PTGS of the RNase H-like protein using a double stranded mRNAof that protein. Average results for three separate experiments of thisnature are depicted in FIG. 20 and show that statistically significantresults are consistently obtained. Overall, these experiments confirmthat SrMV P1 and SrMV HC-Pro inhibits PTGS and that the RNase H-likeprotein is involved in effecting PTGS.

FIG. 21 illustrates the PTGS effect of the RNase H-like protein evenmore clearly. In these figures calli transiently transformed with GUSexhibit the protein. This protein production is severely disrupted bysubsequent or simultaneous transformation with DNA that produces doublestranded GUS mRNA, which triggers PTGS. PTGS is not turned off by thetransformation of the GUS/double stranded GUS calli with RNase H.However, it is severely hampered by introduction of DNA that producesdouble stranded mRNA for RNase H-like protein, which induces PTGS ofRNase H-like protein itself. These experiments demonstrate that theRNase-H like protein plays a role in PTGS. These results are summarizedin Table 4.

TABLE 4 Transformation Constructs Callus Color 35S-int/GUS Blue35S-int/GUS + 35S-int/dsGUS Not Blue 35S-int/GUS + 35S-int/dsGUS + NotBlue 35S-int/RNase H-like protein 35S-int/GUS + 35S-int/dsGUS + Blue35S-int/ds RNase H-like protein

All references mentioned in any portion of the foregoing specificationare incorporated by reference herein.

1. A method of isolating nucleic acid encoding a plant polypeptideactive in PTGS comprising the steps of: i) selecting a bait nucleic acidwhich encodes a bait protein active in PTGS in plants or suppressive ofPTGS in plants; ii) preparing a cDNA prey library from a plant or planttissue wherein the plant or plant tissue actively exhibits PTGS; iii)conducting a yeast two-hybrid assay with the bait and prey nucleicacids, wherein prey cDNA that yields a true positive yeast two-hybridassay result encodes a polypeptide active in PTGS in the plant or planttissue, wherein the bait nucleic acid comprises the sequence ofSEQ.ID.NO. 3 or the sequence of a degenerate code homolog of SEQ.ID.NO.3.
 2. The method of claim 1 further comprising preparing a cDNA preylibrary from a monocot plant.
 3. The method of claim 2 wherein themonocot is selected from the group consisting of sugarcane, corn,sorghum and rice.
 4. The method of claim 1 further comprising the stepof confirming interaction between the bait protein and the polypeptideactive in PTGS using a far-western blot assay.
 5. The method of claim 1further comprising the step of confirming interaction between the baitprotein and the polypeptide active in PTGS using a pull down assay. 6.The method of claim 1 further comprising the step of confirminginteraction between the bait protein and the polypeptide active in PTGSusing an in planta assay.
 7. The method of claim 6 wherein the in plantaassay is conducted in embryonic calli of the plant.
 8. An isolatednucleic acid molecule comprising a nucleic acid molecule having thenucleic acid sequence of SEQ.ID.NO. 3 or the nucleic acid sequence of adegenerate code homolog of SEQ.ID.NO. 3.